Cervical cancers (CaCxs) cause over 250,000 deaths annually are the result of human papillomavirus (HPV) infections. The combination of low HPV vaccine uptake and the average time required for an HPV infection (10-30 years) to cause a CaCx, mean that improving treatment options will remain essential for the foreseeable future. CaCxs require continued HPV oncogene (HPV E6 and E7) expression for initiation and maintenance making these viral oncogenes an attractive target for intervention. Because direct attacking HPV E6 and E7 remains technically difficult, we propose targeting their manipulation of cellular signaling. We identify translesion synthesis (TLS) dysregulation in cervical cancers (CaCx) and show that HPV E6 and E7 cause this aberrant signaling. TLS helps cells tolerate replication stress by preventing replication forks from stalling and collapsing. It exchanges the high fidelity replicative polymerase for a TLS-polymerase (often POL?) that can synthesize DNA with lower fidelity in adverse conditions. HPV E7 causes replication stress by allowing bypass of G1/S-phase checkpoints and depleting nucleoside reserves. This activates TLS, but HPV E6 prevents completion of the pathway by blocking POL? accumulation. We found both HPV oncogenes were capable of preventing POL? induction in response to DNA damaging UV exposure. Both of these phenotypes correlate with increased genome instability and sensitivity to exogenous replication stress (mitomycin C, cisplatin and UV). Our overall hypothesis is that by taxing and inhibiting TLS HPV oncogenes impair genome fidelity in cervical cancers. We test this hypothesis in two related, but distinct specific aims. Aim 1 investigates the destabilizing created by HPV oncogene?s simultaneous activation and inhibition of TLS using primary keratinocytes (the cell type HPV infects) that acutely or constitutively express HPV oncogenes. The mutagenic consequences are defined using whole genome sequencing and established molecular approaches, like immunofluorescence microscopy and immunoblotting. Aim 2 determines the mechanism and consequences of the aberrant TLS response to cisplatin in CaCx. This aim combines specialized approaches (e.g. visualization of metaphase chromosomes) and other molecular techniques. It characterizes TLS in cells after UV and cisplatin exposure, defines the mechanism by which HPV E6 and E7 prevent POL? induction in these cells, and examines the consequences of this TLS hindrance. Undergraduate Student Involvement. Students are the bedrock of this proposal. They were instrumental in generating the corroborative data and will continue to be integral in the proposed experiments. We anticipate preparing 5 undergraduates for futures in research.